The present invention refers to the substantially pure dimer of apolipoprotein AI-Milano (ApoAI-M/ApoAI-M) and pharmaceutical compositions containing this dimer. It also relates to the process for the preparation by recombinant technique as well as by isolation from plasma. The product may be used for the treatment of atherosclerosis and cardiovascular diseases and as a retard formulation of the ApoAI-M
The clear correlation between elevated levels of serum cholesterol and the development of coronary heart disease (CHD) has been repeatedly confirmed, based on epidemiological and longitudinal studies. The definition, however, of complex mechanisms of cholesterol transport in plasma, has allowed the recognition of a selective function of circulating lipoproteins in determining the risk for CHD.
There are, in fact, four major circulating lipoproteins: chylomicrons (CM), very low density (VLDL), low density (LDL) and high density (HDL) lipoproteins. While CM constitute a short-lived product of intestinal fat absorption, VLDL and, particularly, LDL are responsible for cholesterol transport into tissues, including for example the arterial walls. In contrast, HDL are directly involved in the removal of cholesterol from peripheral tissues, carrying it back either to the liver or to other lipoproteins, by a mechanism known as xe2x80x9creverse cholesterol transportxe2x80x9d (RCT).
The xe2x80x9cprotectivexe2x80x9d role of HDL has been confirmed in a number of studies (e.g. Miller et al. Lancet, 1977:965-968 and Whayne et al. Atherosclerosis 1981;39:411-419). In these, the elevated levels of LDL, less so of VLDL, seem to be clearly associated with an increased cardiovascular risk, whereas high HDL levels seem to confer cardiovascular protection. The protective role of HDL has been further strongly supported by the in vivo studies, showing that HDL infusions into rabbits may hinder the development of cholesterol induced arterial lesions (Badimon et al, Lab. Invest. 60, 455-61, 1989)) and/or induce regression of same (Badimon et al, J Clin Invest. 85, 1234-41, 1990).
Recent interest in the study of the protective mechanism/s of HDL has been focussed on apolipoprotein AI (ApoAI), the major component of HDL. High plasma levels of ApoAI are associated with reduced risk of CHD and presence of coronary lesions (Maciejko et al,. N Engl J Med 1983;309:385-389, Sedlis et al,. Circulation 1986;73:978-984).
Plasma ApoAI is a single polypeptide chain of 243 amino acids, whose primary sequence is known (Brewer et al, Biochem Biophys Res Commun 1978;80:623-630). ApoAI is synthesized as a 267 amino acid precursor in the cell. This pre-pro-apoliprotein is processed by N-terminal cleavage first intracellularly where 18 amino acids are lost and then with a further cleavage of 6 amino acids in the plasma or the lymph by the activity of specific proteases.
The major structural requirement of the ApoAI molecule is believed to be the presence of repeat units of 11 or 22 amino acids, presumed to exist in amphipathic helical conformation (Segrest et al, FEBS Lett 1974;38:247-253). This structure allows for the main biological activities of ApoAI, i.e. lipid binding and lecithin cholesterol acyl transferase (LCAT) activation. Another recently described property of ApoAI is its antiviral activity. This has been reported from in vitro studies and is exerted both against Herpes virus strains (Srinivas R V et al, Virology, 1756, 48-57, 1990) and also against the Human Immunodeficiency virus, HIV, (Owe et al,., J Clin Invest, 86, 1142-50, 1990). This activity seems to be exerted by way of an interaction between amphipatic helical portions of ApoAI and envelope glycoproteins of the viruses.
In vitro studies indicate that complexes of ApoAI and lecithin can promote the efflux of free cholesterol from cultured arterial smooth muscle cells (Stein et al,. Ciochem Biophys Acta 1975;380:106-118). By this mechanism HDL can also reduce the proliferation of these cells (Yoshida et al, Exp Mol Pathol 1984;41 :258-266).
More recently, the infusion of ApoAI or of HDL in experimental animals has been shown to exert significant biochemical changes, as well as to reduce the extent and severity of atherosclerotic lesions. After an initial report by Maciejko and Mao (Arteriosclerosis 1982;2:407a), Badimon et al, (see the two quoted studies above) found that they could significantly reduce the extent of atherosclerotic lesions (xe2x88x9245%) and their cholesterol ester content (xe2x88x9258.5%) in cholesterol-fed rabbits, by infusing HDL (d=1.063-1.325 g/ml). They also found that the infusions of HDL led to a close to a 50% regression of established lesions.
It was able to be shown also (Esper et al. Arteriosclerosis 1987;7:523a) that infusions of HDL can markedly change the plasma lipoprotein composition of Watanabe rabbits with inherited hypercholesterolemia, which develop early arterial lesions. In these, HDL infusions can more than double the ratio between the protective HDL and the atherogenic LDL.
The potential of HDL to prevent arterial disease in animal models has been further stimulated by the observation that ApoAI can exert a fibrinolytic activity in vitro (Saku et al, Thromb Res 1985;39:1-8). Ronneberger (Xth Int Congr Pharmacol, Sidney 1987, p 990) demonstrated that extractive ApoAI can increase fibrinolysis in beagle dogs and in Cynomologous monkeys. A similar activity can be noted in vitro on human plasma. This author was able to confirm a reduction of lipid deposition and arterial plaque formation in ApoAI treated animals.
The apolipoprotein AI-Milano (ApoAI-M) is the first described molecular variant of human ApoAI (Franceschini et al, J Clin Invest 1980;66:892-900). It is characterized by the substitution of Arg 173 with Cys (Weisgraber et al, J Biol Chem 1983;258:2508-2513). The mutant apoprotein is transmitted as an autosomal dominant trait and 8 generations of carriers have been identified (Gualandri et al, Am J Hum Genet 1984;37:1083-1097).
The status of the ApoAI-M carrier individual is characterized by a remarkable reduction in HDL-cholesterol level. In spite of this, the affected subjects do not apparently show any increased risk of arterial disease; indeed, by examination of the genealogic tree it appears that these subjects may be xe2x80x9cprotectedxe2x80x9d from atherosclerosis.
The mechanism of the possible protective effect of ApoAI-M in the carriers seems to be linked to a modification in the structure of the mutant apolipoprotein, with the loss of one alpha-helix and an increased exposure of hydrophobic residues (Francheschini et al,. J Biol Chem 1985;260:1632-1635). The loss of the tight structure of the multiple alpha-helices leads to an increased flexibility of the molecule, which associates more readily with lipids, compared to normal AI. Moreover, apolipoprotein/lipid complexes are more susceptible to denaturation, thus suggesting that lipid delivery is also improved in the case of the mutant.
The therapeutic use of the apolipoprotein ApoAI-M mutant is presently limited by the lack of a method allowing the preparation of said apolipoproteins in sufficient amount and in a suitable form.
Another very specific feature of the ApoAI-M, is its capacity to form dimers with itself and complexes with ApoAII, in both cases because of the presence of the Cys residue. From studies of blood fractions containing a mixture of Apolipoproteins, there were indications, showing that the presence of dimers and complexes in the circulation may be responsible for the increased elimination half-life of these in the carriers, recently described in clinical studies (Gregg et al,. NATO ARW on Human Apolipoprotein Mutants: From Gene Structure to Phenotypic Expression, Limone SG, 1988).
ApoAI-M dimers (ApoAI-M/ApoAI-M) act as an inhibiting factor in the interconversion of HDL particles in vitro (Franceschini et al, J Biol Chem 1990;265:12224-12231).
Earlier studies of mixtures containing the dimer have been based on ApoAI-M separated from natural blood from persons with Apo AI-M, which has thus only been obtainable in small quantities.
The difficulty of producing ApoAI and particularly ApoAI-M from plasma fractionation is quite considerable (Franceschini et al, J Biol Chem 1985;260:16321-16325). The isolation and production cannot be done on a big scale, as only a small amount of the raw material is available. Furthermore, there are several risks associated with plasma fractionation products, such as contamination with infectious agents. It is essential that this is avoided.
Attempts have been made to produce human ApoAI, by way of the recombinant DNA technology. In the European patent publication No. 0267703 the preparation of ApoAI from E.coli is described. The process describes a chimeric polypeptide where the ApoAI moiety is fused to the N-terminal amino acid residues of beta-galactosidase or to one or more IgG-binding domains of Protein A, or to the pro sequence of human ApoAI.
The expression of ApoAI and ApoAI-M in yeast strains and the use of the produced components in the treatment of atherosclerosis and cardiovascular diseases is disclosed in WO90/12879. The genes encoding the ApoAI and ApoAI-M were provided with DNA-sequences encoding a yeast-recognizable secretion and processing signals fused upstream to the gene for the mature proteins. A modified MF-alpha-1-leader sequence was used in which the last residues were: HisGlySerLeuAspLysArg.
We have now surprisingly found that the purified dimer Apo AI-M/ApoAI-M has a prolonged plasma half-life compared to the monomer ApoAI-M, further that it has a markedly improved fibrinolysis stimulating property compared to normal ApoAI e.g. its ability to directly activate plasminogen (which normal ApoAI does not), an observation that can be of biological importance, and also the possibility to act as a prodrug for ApoAI-M. It also forms reconstituted HDL (high density lipoprotein) particles of unique size which is not found in recombinant HDL particles containing ApoAI-M or ApoAI.
The present invention relates to substantially pure dimers of apolipoprotein AI-Milano, hereafter called ApoAI-M/ApoAI-M, with a purity of at least 90%, preferably at least 98%, which for the first time has been isolated and characterized from plasma and which also has been produced by recombinant methods. It also relates to pharmaceutical compositions comprising the Apo AI-M/ApoAI-M, optionally together with a stabilizing agent e.g. stabilizing lipid compound such as a phospholipid and/or a carrier.
The pharmaceutical compositions can also contain a lipid lowering agent and/or other medicament already known in the treatment of atherosclerosis and cardiovascular diseases, such as heparin, heparin fractions, and heparin fragments or lipid lowering agents.
Apolipoprotein ApoAI-M can be produced by
a) producing Apolipoprotein AI-Milano by recombinant technology as intracellular fusion protein in E coli, cleaving off Apolipoprotein AI-Milano with formic acid and therefter converting any monomer present to the dimer or
b) producing Apolipoprotein AI-Milano by recombinant technology in which the Apolipoprotein AI-Milano, monomer and dimer, is secreted into the bacterial culture medium in an expression system in E coli and any monomer present thereafter converted to the dimer
and purifying the dimer to a substantially pure form.
According to a) ApoAI-M is produced as a fusion protein intracellularly in the bacteria. The fusion partner is a modified IgG binding domain from protein A, and a cleavage site for formic acid is designed between the fusion partner and ApoAI-M. After lysis of the bacteria, fusion protein was purified on immobilized IgG and cleaved with formic acid. The presence of ApoAI-M and the dimer was showed by Western blotting techniques on a SDS gel electrophoresis.
In example 3 is shown that processed ApoAI-M could be produced in recombinant E. coli and that dimers are formed. However the use of formic acid gives a product truncated with two amino acids in its N-terminal. This truncation is not supposed to alter the activity of the ApoAI-M molecule.
The system according to b) is shown in example 4, in which the completely correct molecule is formed.
The claimed dimer of ApoAI-M can also be obtained
c) by collecting plasma from Apolipoprotein AI-Milano carriers, isolating the HDL apolipoproteins, separating the dimer by use of chromatography, e.g. by Sephacryl chromatography, in several steps or
d) by collecting plasma from Apolipoprotein AI-Milano carriers, purificating the monomer and thereafter converting to the dimer and purifying the dimer to a substantially pure form.
It is important that the separation under c) is effected in several steps and preferably on a long column, such as 300 cm.
If monomer is present, it should always be converted to the dimer form, as shown in the examples below.
The invention includes the method for the treatment of atherosclerosis and cardiovascular diseases and use of the dimer for the preparation of a medicament. The dimer can also be acting as a prodrug for the monomer for the treatment of atherosclerosis and cardiovascular diseases.
This medicament can be used for the treatment of atherosclerosis and cardiovascular diseases and for the prevention and treatment of major cardiocirculatory ailments such as myocardial infarction, unstable angina, acute peripheral vascular occlusions and restenosis after coronary angioplasty.
When chronic arterial conditions are treated, both the coronaries and also peripheral arteries, which are characterized by occlusive plaque, are treated. The dimers will be used for infusion per se with the objective of inducing removal of fat from the plaques or optionally in association with established treatments of atherosclerosis and cardiovascular diseases, such as the use of heparin, heparin fractions and heparin fragments and/or drugs reducing the circulation levels of atherogenic lipoproteins.
The medicament containing the dimer can be used for the prevention and treatment of thrombosis in different clinical circumstances and be used in the stimulation of fibrinolysis.
Amphipathic structures are present to a high extent in the Apo AI-M dimer and the dimer is supposed to have an antiviral effect.
Now for the first time, by the use of the present invention, it has been possible to produce the dimer in an essentially pure form, more than 90% and as much as more than 98% and also to show that this product has a surprisingly better effect on biological systems as compared to ApoAI, which has been shown in our exemples 7-10 below.